Fluorescently labeled growth hormone secretagogue

ABSTRACT

The present invention relates to a fluorescently labeled growth hormone secretagogue which can be used for the identification of compounds capable of binding to growth hormone secretagogue receptors, in particular by high throughput screening.

PRIORITY TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/964,878,filed Oct. 14, 2004 now allowed; which claims the benefit of EuropeanApplication No. 03023568.3, filed Oct. 16, 2003. The entire contents ofthe above-identified applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Growth hormone, which is secreted from the pituitary, stimulates growthof all tissues of the body that are capable of growing. In addition,growth hormone is known to have the following basic effects on themetabolic processes of the body: (1) Increased rate of protein synthesisin all cells of the body; (2) Decreased rate of carbohydrate utilizationin cells of the body; (3) Increased mobilization of free fatty acids anduse of fatty acids for energy. A deficiency in growth hormone secretioncan result in various medical disorders, such as dwarfism.

Methodology is known in the art to determine the activity of a compoundas a growth hormone secretagogue. For example, an ex vivo assay isdescribed by Smith, et al., Science, 260, 1640-1643 (1993) (see text ofFIG. 2 therein), but this assay requires the use of cell cultures anddoes not give an indication of competitive binding activity.Accordingly, it would be desirable to develop a non-radioactivelylabeled ligand which can be used to identify and characterize cellularreceptors which play a role in the activity of growth hormonesecretagogue. It would also be desirable to have a non-radioactivelylabeled ligand available for use in an assay for testing compounds forgrowth hormone secretagogue activity.

Such studies normally require a high specific activity radio-ligand.Previous attempts to develop a binding assay using [T]-labeled or[125I]-labeled peptide ligands derived from GHRP-6 met with limitedsuccess. See R. F. Walker, et al. Neuropharmacol. 989, 28, 1139 and C.Y. Bowers et al., Biochem. Biophys. Res. Comm. 1991, 178, 31 (both ofwhich, to the extent necessary, are herein incorporated by reference).Generally, the binding of such peptide ligands was of low affinity andof excessively high capacity. Moreover, the binding affinities did notcorrelate with the growth hormone secretory activity of the peptides.The lack of correlation of binding and growth hormone secretory activitymost likely was the result of the relatively low specific activity (inthe case of [T] GHRP-6) and non-specific binding properties of theradio-ligands.

These problems are solved by the present invention, which provides afluorescent labeled ghrelin analog (labeled growth hormone secretagogue)which can be used in high throughput screening to identify andcharacterize cellular receptors which play a role in the activity ofgrowth hormone secretagogue and for use in an assay for testingcompounds for growth hormone secretagogue activity.

SUMMARY OF THE INVENTION

The present invention pertains to a labeled growth hormone secretagogueof the formula:R1-Cys-Fwherein R1 is a peptide sequence derived from the ghrelin polypeptidesequence (Seq ID No. 1), and F is a fluorescent dye In a preferredembodiment, R1 is Seq ID No. 2. In another preferred embodiment, R1 isoctanoylated. In another preferred embodiment, R1 is N-octanoylated. Ina more preferred embodiment, R1 is¹Gly-Ser-Dapa(N-octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser.N-octanoylation increases stability of ghrelin towards esteraseactivity. In an even more preferred embodiment, F is selected from thegroup consisting of Texas Red, Tetramethyl rhodamine, MR121. (“Newfluorescent dyes in the red region for biodiagnostics” M. Sauer et al1995 J. Fluoresc. Vol. 5, pp 247-261, or BODIPY-FL4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid). In a most preferred embodiment, F is MR121.

The labeled growth hormone secretagogue herein described can be used foridentifying a compound that can bind to a growth hormone secretagoguereceptor. Said labeled growth hormone secretagogue can also be used toidentify a cellular receptor as a growth hormone secretagogue receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Set-up of HTS assay plate

FIG. 2: Competition curves from Zeiss-System, HTS conditions. A)Competition of ghrelin (1-19) MR121 with H6935; b) Competition ofghrelin (1-19) MR121 with hexarelin.

FIG. 3: Affinity of fluorescent analogs, determined in 125I-ghrelincompetition assays. A) ghrelin (1-19) MR121; b) ghrelin (1-19) TMR; c)ghrelin (1-19) BoFl (BIODIPY-FL); d) ghrelin (1-19) Texas Red.

FIG. 4: Polarization of fluorescent analogs, determined according toprotocol in example 2.2.1 and 2.2.2.

Red (Darker, left hand bar): ghrelin (1-19) MR121, Blue (Lighter, righthand bar): ghrelin (1-19-N-oct) MR121.

Fluorescence polarization of Total bound fluorescent ligands (inpresence of GHSR-1a membranes); 2) Fluorescence polarization of Freeligand (in presence of GHSR-1a membranes and excess of competitor); 3)Fluorescence polarization of fluorescent ligands in buffer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to a labeled growth hormone secretagogueof the formula:R1-Cys-Fwherein R1 is a peptide sequence derived from the ghrelin polypeptidesequence (Seq ID No. 1), and F is a fluorescent dye. In a preferredembodiment, R1 is Seq ID No. 2. In another preferred embodiment, R1 isoctanoylated. In another preferred embodiment, R1 is N-octanoylated. Ina more preferred embodiment, R1 is¹Gly-Ser-Dapa(N-octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser.N-octanoylation increases stability of ghrelin towards esteraseactivity. In an even more preferred embodiment, F is selected from thegroup consisting of Texas Red, Tetramethyl rhodamine, MR121. (“Newfluorescent dyes in the red region for biodiagnostics” M. Sauer et al1995 J. Fluoresc. Vol. 5, pp 247-261, or BODIPY-FL4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid). In a most preferred embodiment, F is MR121.

The present invention also provides a process of synthesizing a labeledghrelin which comprises the steps of a) coupling a Cys to the C-terminalamino acid of ghrelin; and b) reacting the thiol-containing ghrelin to afluorescent dye. Preferably, said fluorescent dye is selected from thegroup consisting of Texas Red, Tetramethyl rhodamine, MR121. (“Newfluorescent dyes in the red region for biodiagnostics” M. Sauer et al1995 J. Fluoresc. Vol. 5, pp 247-261, or BODIPY-FL(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid).

The present invention also pertains to a method for identifying acompound that can bind to a growth hormone secretagogue receptorcomprising contacting said compound (a “test” compound) with a hostexpressing a growth hormone secretagogue receptor in the presence of thelabeled growth hormone secretagogue hereinbefore described, or with amembrane preparation from such a host, and monitoring whether thecompound influences the binding of the labeled growth hormonesecretagogue hereinbefore described to the growth hormone secretagoguereceptor by measuring the fluorescence of the label of the bound labeledgrowth hormone secretagogue hereinbefore described.

The host may be a tissue sample, primary cells or cultured cells whicheither naturally expresses a growth hormone secretagogue receptor, orwhich are either transiently or stably transfected with a growth hormonesecretagogue receptor. Methods of transfecting cells are well known inthe art (Sambrook et al., Molecular Cloning: A Laboratory Manual (1989),Cold Spring Harbor Laboratory Press, New York, USA).

Preferably, said method is a high throughput screening method. Themethod hereinbefore described is sensitive with regard to the pH of theassay buffer used. A pH deviation of 0.2 results in a 50% loss ofbinding. Therefore, in a preferred embodiment, the assay buffer used insaid method has a pH of 7.2. The peptides of this invention tend toabsorb to surfaces. Thus, in a preferred embodiment of this invention,the plastic ware either comprises a nonbinding surface or is blockedwith a casein solution. The term “plastic ware” as used herein refers toplates used for the assay, as well as any type of tube used to preparesolutions comprising the peptides of this invention. Preferably, saidcasein solution comprises 1% casein. More preferably, blocking isperformed over night. In a most preferred embodiment, the blocked platesare washed with buffer before use.

The present invention also provides a method of identifying a cellularreceptor as a growth hormone secretagogue receptor comprising contactinga host suspected to express a growth hormone secretagogue receptor withthe labeled growth hormone secretagogue hereinbefore described anddetermining whether binding has occurred.

Furthermore, the present invention pertains to a method for identifyingthe activity of a compound (“test compound”) as a growth hormonesecretagogue comprising contacting the compound suspected of havingactivity as a growth hormone secretagogue with a host expressing agrowth hormone secretagogue receptor in the presence of the labeledgrowth hormone secretagogue hereinbefore described and monitoringwhether the compound suspected of having activity as a growth hormonesecretagogue influences the binding of the labeled growth hormonesecretagogue hereinbefore described to the growth hormone secretagoguereceptor.

The present invention also provides a compound identified by methodshereinbefore described or pharmaceutically acceptable salts thereof. Inaddition, the present invention provides a pharmaceutical compositioncomprising a compound hereinbefore described and a pharmaceuticallyacceptable carrier.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the identified agents wherein the parent agent is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,benzenesulfonic, toluenesulfonic, methanesulfonic, ethane disulfonic,oxalic, isethionic, and the like.

The term “secretagogue” means a substance that stimulates the secretionof a hormone, specifically growth hormone.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent agent which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

The agents identified by the method of the invention may be modified toachieve (i) modified site of action, spectrum of activity, and/or (ii)improved potency, and/or (iii) decreased toxicity (improved therapeuticindex), and/or (iv) decreased side effects, and/or (v) modified onset ofaction, duration of effect, and/or (vi) modified kinetic parameters(resorption, distribution, metabolism and excretion), and/or (vii)modified physico-chemical parameters (solubility, hygroscopicity, color,taste, odor, stability, state), and/or (viii) improved generalspecificity, organ/tissue specificity, and/or (ix) optimized applicationform and route by (i) esterification of carboxyl groups, or (ii)esterification of hydroxyl groups with carbon acids, or (iii)esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates orsulfates or hemi succinates, or (iv) formation of pharmaceuticallyacceptable salts, or (v) formation of pharmaceutically acceptablecomplexes, or (vi) synthesis of pharmacologically active polymers, or(vii) introduction of hydrophilic moieties, or (viii)introduction/exchange of substituents on aromates or side chains, changeof substituent pattern, or (ix) modification by introduction ofisosteric or bioisosteric moieties, or (x) synthesis of homologouscompounds, or (xi) introduction of branched side chains, or (xii)conversion of alkyl substituents to cyclic analogues, or (xiii)derivatisation of hydroxyl group to ketales, acetates, or (xiv)N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannichbases, imines, or (xvi) transformation of ketones or aldehydes toSchiff's bases, oximes, acetates, ketales, enolesters, oxazolidines,thiozolidines or combinations thereof; and (b) formulating the productof said modification with a pharmaceutically acceptable carrier or acarrier/diluent acceptable for fragrance or flavor compositions orproducts.

Any conventional carrier material can be utilized. The carrier materialcan be an organic or inorganic one suitable for eteral, percutaneous orparenteral administration. Suitable carriers include water, gelatin, gumarabic, lactose, starch, magnesium stearate, talc, vegetable oils,polyalkylene-glycols, petroleum jelly and the like. Furthermore, thepharmaceutical preparations may contain other pharmaceutically activeagents. Additional additives such as flavoring agents, stabilizers,emulsifying agents, buffers and the like may be added in accordance withaccepted practices of pharmaceutical compounding.

The present invention also pertains to the labeled ligand, compounds,methods, process, uses and composition substantially as hereinbeforedescribed, especially with reference to the following examples.

General Synthesis Schemes

Fluorescent Labeling

In general, the covalent labeling of a cystein containing peptide withmaleimide conjugated fluorophore can be accomplished as described below.

A thiol-containing peptide is dissolved in 9:1 DMSO: 50 mM phosphatebuffer pH 6 to a final concentration of 1-10 mM. Fluorophore reagent(10-50 mM freshly prepared solution in DMSO) is added stepwise and themixture left to react at room temperature for a bout 10 minutes. Theprogress of the reaction is followed by analysis of samples, for examplewith RP-HPLC. Upon completion, the fluorescent labeled peptide isisolated, for example by RP-HPLC.

Coupling of Cysteine to C-Terminus of Peptide

This synthetic coupling procedure is a standard procedure of peptidesynthesis and is known to those skilled in the art. One such procedurethat may be used is provided in Chan and White, Fmoc Solid Phase PeptideSynthesis: A Practical Approach (Oxford University Press 2000).

EXAMPLES

The following examples are provided for illustrative purposes and arenot intended to limit the scope of applicants' invention.

Example 1 Synthesis of the Labeled Peptides Example 1.1 Synthesis of¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys-NH₂

Continuous-flow solid-phase synthesis was performed on a Pioneer™Peptide Synthesis System, starting from Tenta Gel S RAM resin (0.25mmol/g (Rapp Polymere GmbH, Tübingen, Germany) according to the methoddescribed by Chan and White, Fmoc Solid Phase Peptide Synthesis: APractical Approach, pp. 41-74 (Oxford University Press 2000). Thebase-labile Fmoc group (Sygena) was used for α-amino protection. Sidechains were protected with the following protection groups: -Asn(Trt),Glu(OtBu), Ser(tBu), His(Trt), Gln(Trt), His(Trt), Arg(Pbf), Lys(Boc),Cys(Trt). Fmoc-amino acids (4 equiv., Novabiochem) were activated withan equivalent amount ofO-(1,2-dihydro-2-oxopyrid-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU, Fluka) and N,N-diisopropylethylamine (Hünig'sbase, Acros). Fmoc deprotection was achieved with 20% piperidine (Fluka)in DMF (Fluka). The automated synthesis was interrupted after residue⁴Phe was incorporated in the target sequence. Peptide synthesis wascontinued semi-manually using a Peptide Synthesizer SP650 (Labortec AG).Side chain unprotected Fmoc-³Ser-OH (0.65 g, 2 mmol, Fluka), TPTU (0.59g, 2 mmol), Hünig's base (1.03 ml) were added to the peptide resin andcoupling was continued for 1 hour in DMF solvent (ninhydrin negative).Octanoylation of the side chain hydroxyl was achieved using caprylicacid (2.0 ml, 12 mmol, Fluka), N,N′-Diisopropylcarbodiimide, (1.9 ml, 12mmol, Fluka) N,N′-dimethylaminopyridine (18 mg, 0.15 mmol, Fluka) inN-methylpyrrolidone (Fluka) solvent. After 4 hours the reaction mixturewas filtered off and synthesis was continued using the standard peptidesynthesis protocol (above).¹Gly-Ser(tBu)-Ser(octanoyl)-Phe-⁵Leu-Ser(tBu)-Pro-Glu(OtBu)-His(Trt)-¹⁰Gln(Trt)-Arg(Pbf)-Val-Gln(Trt)-Gln(Trt)-¹⁵Arg(Pbf)-Lys(Boc)-Glu(OtBu)-Ser(tBu)-Cys(Trt)-NH₂Tenta Gel S-resin (2.0 g) was treated with a mixture (100 ml) of 95%TFA, 2.5% H₂O, 2.5% EDT, 2.5% triisopropylsilane for 5 hours. Thereaction mixture was concentrated and poured into diethyl ether and theprecipitate was collected by filtration and lyophilized from water. Thecrude peptide (0.80 g) was purified by preparative RP-HPLC. There wasobtained¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys-NH₂(0.18 g, Ion-spray MS analysis (M+2H)²⁺/2=1165.4, (M+3H)³⁺/3=777.2).

Example 1.1b Synthesis of¹Gly-Ser-Dapa(N-octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys-NH₂(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-octanoylamino-propionicacid; Fmoc-L-Dapa(N-octanoyl)-OH

To a pre-activated mixture containing caprylic acid (1.54 ml, 10 mmol),TPTU (2.8 g, 9.5 mmol) and Hunig's base (3.4 ml, 20 mmol)) in DMF (20ml) was added a solution of Fmoc-L-Dapa-OH Neosysytem FA04002 8 (3.3 g,10 mmol) in DMF (10 ml). The reaction mixture was stirred for 1 h,concentrated under reduced pressure dissolved in ethyl acetate andwashed with 5%/10% KHSO₄/K₂SO₄, brine, dried over Na₂SO₄, filtered andconcentrated. Crystallizaton from ethyl acetate/hexane: 3.7 g, 82%;MS=451.4 (MH)⁻.

The peptide synthesis incorporating Fmoc-L-Dapa(N-octanoyl)-OH wasperformed on a Pioneer™ Peptide Synthesis System as described abovestarting with Tentagel S-NH2 resin (0.55 mmol), yielding purifiedpeptide ¹Gly-Ser-Dapa(N-octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys-NH₂: 135mg; Ion-spray MS: (M+2H)²⁺/2=1164.8, (M+3H)³⁺/3=776.8).

Conjugation of Peptides to Fluorophores Example 1.2¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys(TxR)-NH₂

The thiol-containing peptide above (1.3 mg) was dissolved in 9:1 DMSO:50 mM phosphate buffer pH 6 to a final concentration of 2.5 mM. 1.2equivalent of TxR (Texas Red)-Maleimide (30 mM freshly prepared solutionin DMSO, from Molecular Probes, Leiden, Netherlands, Product No.#T-6008) was added and the mixture left to react at room temperature for10 minutes. The reaction mixture was directly purified by RP-HPLC: 0.17mg. MS analysis: calculated monoisotopic mass: C136H198N36O39S3=3055.38;found monoisotopic mass: 3055.35

Example 1.3¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys(TMR)-NH₂

The TMR (Tetramethylrhodamine) derivatized peptide (Molecular Probes,Leiden, Netherlands, Product No. #T-6027) was prepared analogously toexample 1.2: From 1.7 mg starting peptide material was isolated 0.44 mglabeled peptide. MS analysis: calculated monoisotopic mass:C127H185N35O36S=2808.34; found monoisotopic mass: 2808.40

Example 1.4¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys(MR121)-NH₂

The MR121* derivatized peptide was prepared analogously to example 2:From 1.5 mg initial peptide material was isolated 0.37 mg labeledpeptide. MS analysis: calculated monoisotopic mass:C129H196N37O35S=2855.44; found monoisotopic mass: 2855.38

Example 1.5¹Gly-Ser-Ser(octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys(BODIPY-FL)-NH₂

The BODIPY-FL(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid, from Molecular Probes, Leiden, Netherlands, Product No. #B-10250)derivatized peptide was prepared analogously to example 2: From 0.7 mgstarting material was isolated 0.20 mg labeled peptide. MS analysiscalculated monoisotopic mass: C119H183BF2N36O34S=2742.4; foundmonoisotopic mass: 2742.4.

Example 1.6¹Gly-Ser-Dapa(N-octanoyl)-Phe-⁵Leu-Ser-Pro-Glu-His-¹⁰Gln-Arg-Val-Gln-Gln-¹⁵Arg-Lys-Glu-Ser-Cys(MR121)-NH₂

The MR121* derivatized peptide was prepared analogously to example 2:From 0.7 mg initial peptide material was isolated 0.17 mg labeledpeptide. MS analysis: calculated monoisotopic mass:C129H197N36O35S=2854.46; found monoisotopic mass: 2854.49. *MR-121 is anoxazine fluorescent dye. [see lit.: “New fluorescent dyes in the redregion for biodiagnostics” M. Sauer et al 1995 J. Fluoresc. Vol. 5, pp247-261]; Reference for MR121: Marmé et al., Bioconjugate Chem. 2003,14, 1133-1139.

Example 2 Binding Assay Example 2.1 Membrane Preparation

Human Embryonic Kidney HEK 293 (EBNA) cells were grown in suspension andtransfected according to the method previously described (Schlaeger andChristensen, Cytotechnology, 30, 71-83, 1999). The cells werecentrifugated for 10 min at 500 rpm, washed once with PBS-0.7 mMEDTA/(4° C.) and resuspended in PBS-EDTA-PI (with Protease inhibitorcocktail), at 2 ml/g of cells. Cells were broken with Ultra Turax levelgreen 3×15″ with 30″ breaks on ice. To remove debris the suspension wascentrifugated in a Sorvall SS34 rotor for 20 min at 2000 rpm. Thesupernatant was collected and centrifugated for 40 min at 20'000 rpm.The pellet was resuspended in PBS-EDTA. Receptor density was verifiedwith saturation binding assay using T-MK 0677 to be 4.9 pmol/mg protein.

Example 2.2 FP-Assay Example 2.2.1 Assay Development

GHSR-1a-containing cell-membranes was diluted in FP-buffer: 25 mM Hepes,5 mM MgCl₂, 1 mM CaCl₂, 4% PEG, 0.1% BSA (fraction V) to a to a finalvolume of 0.5-1 ml, passed through a 0.4 mm syringe and sonicated 4times 20 pulses while kept on ice. 10 nM solution of MR121-labeledghrelin (tracer) and 20× concentrated solutions of competitor inFP-buffer were prepared. To determine polarization of receptor boundtracer, three samples of 180 ul were prepared: Total bound:Membranes+tracer, Free ligand: membranes+tracer+competitor, Fluorescencebackground: membranes+buffer. 3×50 μl of each sample was transferred toassay-plates right after mixing.

Example 2.2.2 HTS Protocol for FP-Ghrelin Competition Binding Assay

FIG. 1. shows a possible layout of a 384 well plate for High ThroughputScreening.

For one round of screening, 119 Sample Plates and 1 DMSO Plate(membranes p20F1+p22F1) were used. The following is a representativenon-limiting example of a HTS protocol with MR121 as fluorochrome. Forthe assay, Costar 384 well UV plates with non-binding surface were used.The following assay buffer was used: 25 mM Hepes pH 7.2, 5 mM MgCl₂, 1mM CaCl₂, 4% polyethylene glycol, and 0.1% BSA (Fract. V) was addedfresh every week. Receptors were provided as follows: Membranes wereisolated as described in example 2.1. Final assay concentration was 1.4nM of GHSR-1a as determined by 125 I-ghrelin saturation binding. Typicalvalues for the membrane stock are: Bmax=6 fmol/μg; ProteinConcentration=50 μg/μl.

To avoid sedimentation membranes need to be pushed through a needle(3×/0.4 mm) and sonicated “Branson sonifier 250” set to intensity level3-4, 4×20 pulses separated by 30 sec pause. Membranes were kept on iceduring sonication. Membranes were diluted to an endconcentration of 1.4nM of receptor.

The tracer ghrelin(1-19) [K19MR121] was diluted in buffer from 1 μM DMSOstock. Final assay concentration was 0.5 nM. Since the peptide tends toadsorb to surfaces, the plastic ware used for the diluted peptidesolution either comprised a nonbinding surface or was blocked overnightwith a 1% casein solution, and then washed with buffer before use.

The following steps were used for high throughput screening:

-   1) 30 μl of 1.33× membrane solution were added to all wells of the    assay plate.-   2) 4.4 μl of buffer were transferred to “FPBLK”/“100% control” wells    and 4.4 μl of reference compound solution were transferred to “STD”    and “0% control” wells from a reservoir plate to the assay plate-   3) 10 μl of water with 0% DMSO were added to columns 3 to 24 of the    compound storage plate containing 1 μl of 2 mM compounds (Endconc.    20 μM).-   4) The contents of the storage plate were mixed.-   5) 4.4 μl of diluted compounds were transferred from the storage    plate to an assay plate.-   6) The contents of the assay plate were mixed five times and then    incubated for 30 min at 24° C.-   7) 5.6 μl of 7.143× tracer solution were added to the assay plate    except to wells A1 to D2 (wells labeled “FPBLK” in FIG. 1) to which    5.6 μl of buffer were added.-   8) The content of the assay plate was mixed five times.-   9) 30 μl of the solution in each well were transferred from the    assay plate to the read-out plate (Corning UV non-binding surface).-   10) The readout plate was incubated for 10 min. at RT.-   11) MR121 fluorescence was read from the read-out plate at 650-695    nm (5 s, Focus To Bottom 1 mm, xy scan 0.5 mm), with settings at    parallel (∥) and crossed (⊥) polarization, using a Zeiss    plate::vision microtiter plate reader.

1. A method of identifying a cellular receptor as a growth hormonesecretagogue receptor comprising (a) contacting a host suspected toexpress a growth hormone secretagogue receptor with a labeled growthhormone secretagogue of the formulaR₁-Cys-F wherein R₁ is a peptide sequence derived from the ghrelinpolypeptide sequence of SEQ ID NO. 2 and F is a fluorochrome, and, (b)determining whether binding has occurred.
 2. A method for identifying acompound that can bind to a growth hormone secretagogue receptorcomprising the steps of a) contacting said compound with a membraneisolated from a host expressing a growth hormone secretagogue receptorin the presence of the labeled growth hormone secretagogue of claim 1 inan assay buffer; b) washing said membrane to remove unbound labeledgrowth hormone secretagogue; and c) monitoring whether the compoundinfluences the binding of the labeled growth hormone secretagogue ofclaim 1 to the growth hormone secretagogue receptor by measuring thefluorescence of said membranes.
 3. The method of claim 2, wherein saidmethod is a high throughput screening method.
 4. The method of claim 2,wherein said assay buffer has a pH of 7.2.
 5. A method for identifying acompound that has growth hormone secretagogue activity comprising (a)contacting the test compound with a host expressing a growth hormonesecretagogue receptor in the presence of the labeled growth hormonesecretagogue of claim 1 and (b) determining whether the test compoundinfluences the binding of the labeled growth hormone secretagogue ofclaim 1 to the growth hormone secretagogue receptor.